Angiogenesis is the formation of new capillary blood vessels as outgrowths of pre-existing vessels. The tightly regulated process plays a vital role in many physiological processes, such as embryogenesis, wound healing and menstruation. Angiogenesis is also important in certain pathological events. In addition to a role in solid tumor growth and metastasis, other notable conditions with an angiogenic component are arthritis, psoriasis and diabetic retinopathy (Hanahan and Folkman (1996) Cell 86:353–364; Fidler and Ellis (1994) Cell 79(2):185–188).
At the onset of angiogenesis, the quiescent endothelium is destabilized into migratory, proliferative endothelial cells. The angiogenic (activated) endothelium is maintained primarily by positive regulatory molecules. In the absence of such molecules, the endothelium remains in a differentiated, quiescent state that is maintained by negative regulatory molecules, angiogenesis inhibitors (Bouck (1990) Cancer Cells 2:179–185; Hanahan and Folkman (1996) supra). Normally, the negative and positive activities are balanced to maintain the vascular endothelium in quiescence (Hanahan and Folkman (1996) supra; Folkman and Klagsbrum (1987) Science 235:442–447). A shift in the balance of the positive and negative regulatory molecules can alter the differentiated state of the endothelium from the non-angiogenic, quiescent to the angiogenic state (Hanahan and Folkman (1996) supra). In the switch to pro-angiogenesis, the quiescent endothelial cells are stimulated to migrate toward a chemotactic stimulus, lining up in a tube (sprout) formation (Folkman and Klagsbrum (1987) supra). These cells also secrete proteolytic enzymes that degrade the endothelial basement membrane, thus allowing the migrating endothelial cells to extend into the perivascular stroma to begin a new capillary sprout. The angiogenic process is characterized by increased proliferation of endothelial cells to form the extending capillary (Folkman and Klagsbrum (1987) supra; Moses, et al. (1995) Int. Rev. Cytol. 161:1–48; Martiny-Baron and Marme (1995) Curr. Opin. Biotechnol. 6:675–680; Liotta, et al. (1991) Cell 64:327–336).
Vascular endothelial growth factor (VEGF) is a mitogenic factor that stimulates pro-angiogenic properties, including endothelial cell migration and proliferation. VEGF induces the expression of plasminogen activator proteolytic pathway proteins that participate in cellular invasive and remodeling processes (Pepper, et al. (1991) Biochem. Biophys. Res. Commun. 181:902–906; Mandriota, et al. (1995) J. Biol. Chem. 270:9709–9716; Mignatti, et al. (1989) J. Cell. Biol. 108:671–682). VEGF-A RNA can undergo alternative splicing to produce four isoforms (Leung, et al. (1989) Science 246:1306–1309; Houck, et al. (1991) Mol. Endocriniology 5:1806–1814; Tischer, et al. (1991) J. Biol. Chem. 26611947-22954). Three of those isoforms, VEGF-A165,189,206, bind to heparin. Pro-VEGF affinity for heparin appears to be important in the regulation of the availability of VEGF at the cell surface (Houck, et al. (1992) J. Biol. Chem. 267:26031–26037), where it can interact with its tyrosine kinase receptors to exert its activity (Gitay-Goren, et al. (1992) J. Biol. Chem. 267:6093–6098). VEGF-A can be released from heparin in an inactive or active form (Ortega, et al. (1998) Biol. Cell 90:381–390). Plasmin and urokinase plasminogen activator (uPA) cleaves pro-VEGF into an active form of varied sizes depending upon the isoform and the activator molecule (Plouet, et al. (1997) J. Biol. Chem. 272:13390–13396).
There are naturally occurring molecules that serve as negative regulators of angiogenesis. Angiostatin, one such negative regulator, is a 38–45 kDa cleavage product of plasminogen, containing kringle domains 1–4 (K1–4) (O'Reilly, et al. (1994) Cell 79:315–328; O'Reilly, et al. (1996) Nat. Med. 2:689–692). Plasminogen, the precursor of plasmin, is activated when it is cleaved at the carboxy-terminus by plasminogen activators. The amino terminus contains five consecutive kringle domains, each approximately 9 kDa. The greatest inhibitory activity of angiostatin is contained within kringles 1–3 (Cao, et al. (1996) J. Biol. Chem. 271:29461–29467) and kringles 1–5 (Cao (1999) Proc. Natl. Acad. Sci. USA 6:5728–5733). The mechanism for angiostatin inhibition of endothelial cell growth in vitro and angiogenesis in vivo is unclear.
Plasminogen activator inhibitor-1 (PAI-1), a serpin family, serine protease inhibitor, is a multifunctional regulatory protein in the plasminogen activator proteolytic (Chapman, et al. (1982) Cell 28:653–662; Chapman (1997) Curr. Opin. Cell Biol. 9:714–724) and fibrinolytic pathways (Loskutoff and Curriden (1990) Ann. NY Acad. Sci. 598:238–247; Collen (1999) Thromb. Haemost. 82:258–270). Active PAI-1 (vitronectin-bound) inhibits proteolytic degradation of the extracellular matrix by inhibiting uPA/tPA, which in turn inhibits cell migration and invasion (Blasi (1999) Thromb. Haemost. 82:298–304). PAI-1 can exist in an active, inactive/latent or substrate-cleaved conformation (Lawrence, et al. (1997) J. Biol. Chem. 272:7676–7680; Debrock and Declerck (1998) Thromb. Haemost. 79:597–601). The PAI-1 reactive center loop (RCL), located at amino acids 320–351 (Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27:157–162; Laskowski and Kato (1980) Annu. Rev. Biochem. 49:593–626), initially interacts with uPA at Arg-346 (Lawrence, et al. (1994) J. Biol. Chem. 269:27657–27662; Tucker and Gerard (1996) Eur. J. Biochem. 237:180–187) to form a stable PAI-1/uPA complex to inactivate uPA (York, et al. (1991) J. Biol. Chem. 266:8495–8500). In the active/latent configuration of PAI-1 (not bound to vitronectin), the RCL spontaneously inserts into the β-sheet of strand 4a to stabilize the PAI-1 structure (Mottonen, et al. (1992) Nature 355:270–273; Egelund, et al. (1997) Eur. J. Biochem. 248:775–785; Kjoller, et al. (1996) Eur. J. Biochem. 241:38–46). It has been shown that when PAI-1 is cleaved between residues P and P′ in the RCL, PAI-1 is converted to a substrate (Lawrence, et al. (1997) supra; Debrock and Declerck (1998) supra). In the cleaved conformation, the RCL is partially inserted into β-sheet of strand A, thus making the structure of cleaved and inactive PAI-1 more similar to each other than to active PAI-1. However, it has been demonstrated that there are distinct conformational differences between latent and cleaved PAI-1 (Sancho (1995) Biochemistry 34:1064–1069). The PAI-1 region distant from the RCL contains many binding domains for regulatory molecules involved in the proteolytic and fibrinolytic pathways. This region of PAI-1 has interactive sites for vitronectin (Lawrence, et al. (1994) supra; Padmanabhan and Sane (1995) Thromb. Haemost. 73:829–834; Van Meijer, et al. (1994) FEBS Lett. 352:342–346; Seiffert, et al. (1994) J. Biol. Chem. 269:2659–2666), heparin (Ehrlich, et al. (1992) J. Biol. Chem. 267:11606–11611), tPA, uPA (Keijer, et al. (1991) Blood 78:401–409; Reilly and Hutzelmann (1992) J. Biol. Chem. 267:17128–17135), thrombin (Ehrlich, et al. (1992) supra), and fibrin (Ehrlich, et al. (1992) supra; Reilly and Hutzelmann (1992) supra). Through its interactions with some of the same regulatory molecules in the proteolytic and fibrinolytic pathways, it has been demonstrated that PAI-1 (active and inactive) is also able to play a role in anti-angiogenic mechanisms (Mulligan-Kehoe, et al. (2001) J. Biol. Chem. 276:8588–8596; Schnaper, et al. (1995) J. Cell Physiol. 165:107–118; Stefansson, et al. (2001) J. Biol. Chem. 276:8135–8141).
A recent report demonstrates that when a truncated porcine PAI-1 protein rPAI-123, is incubated with plasminogen and uPA, it induces formation of an angiostatin-like protein that has proteolytic activity (Mulligan-Kehoe, et al. (2001) supra). In this reaction, angiostatin is formed from cleaved plasmin. uPA enhances the formation of the angiostatin-like protein by increasing the amount of available plasmin. The proteolytic activity of the 36 kDa angiostatin is ultimately inhibited by increasing amounts of rPAI-123 that are available for binding uPA and/or plasminogen. In this second mechanism, rPAI-123 reduces the numbers of uPA/plasminogen interactions; thus, reducing the amount of plasmin produced. Cultured endothelial cells exposed to rPAI-123 exhibit a decrease in proliferation, increased apoptosis, and decreased migration in the presence of VEGF. This truncated PAI-1 appears to be exposing sites that participate in a functional role for PAI-1 in generating angiostatin fragments from plasmin.
Previously, zymographic analysis demonstrated the importance of rPAI-123 interactions with uPA, plasminogen, and plasmin that result in angiostatin formation. Furthermore, it has been shown that rPAI-123 blocks migration of VEGF-stimulated endothelial cells (Mulligan-Kehoe, et al. (2001) supra).
Anti-angiogenic tumor treatment strategies are based upon inhibiting the proliferation of budding vessels, generally at the periphery of a solid tumor. These therapies are often applied to reduce the risk of micrometastasis or to inhibit further growth of a solid tumor after more conventional intervention (such as surgery or chemotherapy).
The recognition of VEGF as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been proposed for use in interfering with VEGF signaling (Siemeister et al. (1998) Cancer Metastasis Rev., 17(2):241–248). Monoclonal antibodies against VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (Kim, et al. (1993) Nature 362:841–844; Asano, et al. (1998) Hybridoma 17:185–90; Mesiano, et al. (1998) Am. J. Pathol. 153(4):1249–1256; Luo, et al. (1998) Cancer Res. 58(12):2594–2600; Borgstrom, et al. (1996) Prostate 35(1):1–10; Borgstrom, et al. (1998) Anticancer Research 19(5B):4203–11). Moreover, U.S. Pat. No. 6,342,221 to Thorpe, et al. discloses the use of anti-VEGF antibodies to specifically inhibit VEGF binding to the VEGFR-2 receptor.
Regulation of angiogenesis by PAI proteins has also been discussed. U.S. Pat. No. 5,830,880 to Sedlacek, et al. discloses the expression of PAI-1, PAI-2, PAI-3 and angiostatin through a gene therapy approach to inhibit angiogenesis. Furthermore, RCL mutants (residues 331–346) of PAI-1 have been disclosed in PCT Publication No. WO 97/39028 which are resistant to elastase inactivation and/or have a high affinity for vitronectin.